A key player in mast cell activation is the high affinity IgE receptor, Fc5RI. While much is known about the chain of events initiated by crosslinking of Fc?RI through multivalent antigen, there is a fundamental gap in our understanding of how protein dynamics facilitate signaling. Our long term goal is to understand how the dynamic and stochastic behavior of protein-protein interactions influences signal propagation. The objective of this proposal is to quantify dynamic protein interactions during early events in Fc?RI signaling. Our central hypothesis is that the duration of protein-protein interactions modulates the signaling outcome. The rationale for the proposed research is that understanding the role of protein-protein interaction dynamics in signaling is the next step in understanding how the cell shapes the strength and quality of an immune response. We will integrate multiple imaging modalities, both novel and established, to capture and quantify early events that govern Fc?RI membrane associated signaling in order to achieve three specific aims: 1) To determine the interaction lifetime and diffusional dynamics that facilitate Fc?RI activation; 2) To quantify the kinetics of Syk adaptor protein binding to the signaling complex; and 3) To determine the cluster stability of LAT and the interplay between Fc?RI and LAT signaling patches. The approach is innovative because we will develop methodology for imaging biochemical events at the molecular level that will allow us to obtain dynamic information about signaling events that cannot be determined using traditional biochemistry techniques. The proposed research is significant because the quantitative information that we propose to obtain has not been directly measured before and will bring new perspectives to the cell biology community. Furthermore, the development of multi-color single molecule imaging techniques described here will be applicable to many other fundamental biological questions. The information we gain about Fc5RI signaling will help to fill the gaps in our knowledge of how Fc5RI initiates signaling. Ultimately, we expect that this information will open new avenues for drug design that target protein interactions and localization.